The enzyme ADP-ribose transferase (ADPRT) (E.C.4.2.30) is a chromatin-bound enzyme located in the nucleus of most eukaryotic cells. The enzyme catalyzes the polymerization of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD.sup.+) to form poly (ADP-ribose). The polymer is covalently attached to various nuclear proteins, including the polymerase itself.
The many varied roles that ADP ribosylation plays in cellular metabolism have made ADPRT a target for drugs essentially useful for combating neoplasia and viral infections. Numerous physiological activities have been detected for compounds that inhibit the polymerase activity of ADPRT. Such activities include a cell cycle dependent prevention of carcinogen-induced malignant transformation of human fibroblasts (Kun, E., Kirsten, E., Milo, G. E. Kurian, P. and Kumari, H. L. (1983) Proc. Natl. Acad. Sci. USA 80:7219-7223), conferring also carcinogen resistance (Milo, G. E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Lett. 179.:332-336), inhibition of malignant transformation in hamster embryo and mouse C3H10T1/2 cell cultures (Borek, C., Morgan, W. F., Ong, A. and Cleaver, J. E. (1984) Proc. Natl. Acad. Sci. USA 81:243-247), deletion of transfected oncogenes from NIH 3T3 cells (Nakayashu, M., Shima, H., Aonuma, S., Nakagama, H., Nagao. M. and Sugimara, T. (1988) Proc. Natl. Acad. Sci. USA 85:9066-9070), suppression of the mitogenic stimulation of tumor promoters (Romano, F., Menapace, L. and Armato, V. (1983) Carcinogenesis 9:2147-2154), inhibition of illegitimate DNA recombinations (Waldman, B. C. and Waldman, A. (1990) Nucl. Acids Res. 18:5981-5988) and integration (Farzaneh, F., Panayotou, G. N., Bowler, L. D., Hardas, B. D., Broom, T., Walther, C. and Shall, S. (1988) Nucl. Acids Res. 16: 11319-11326), induction of sister chromatid exchange (Ikushima, T. (1990) Chromosoma 99:360-364) and the loss of certain amplified oncogenes (Grosso, L. E. and Pitot, H. C. (1984) Biochem. Biophys. Res. Commun. 119:473-480; Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci. USA 86:7442-7445).
Compounds known to inhibit ADPRT polymerase activity include benzamide (Kun, E., Kirsten, E., Milo, G. E. Kurian, P. and Kumari, H. L. (1983) Proc. Natl. Acad. Sci. USA 80:7219-7223), substituted benzamides (Borek, C., Morgan, W. F., Ong, A. and Cleaver, J .E. (1984) Proc. Natl. Acad. Sci. USA 81:243-247; Romano, F., Menapace, L. and Armato, V. (1983) Carcinogenesis 9; 2147-2154; Farzaneh, F., Panayotou, G. N., Bowler, L. D., Hardas, B. D., Broom, T., Walther, C. and Shall, S. (1988) Nucl. Acids Res. 16:11319-11326.; Grosso, L. E. and Pitot, H. C. (1984) Biochem. Biophys. Res. Commun. 119:473-480; Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci. USA 86:7442-7445), 3-aminonaphthylhydrazide (Waldman, B. C. and Waldman, A. (1990) Nucl. Acids Res. 18:5981-5988), isoquinoline, quercetin, and coumarin (1,2-benzopyrone) (Milo, G. E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Lett. 179: 332-336). The anti-transforming and anti-neoplastic effect of 1,2 benzopyrone were demonstrated in vitro and in vivo (Tseng, et al., (1987) Proc. Natl. Acad. Sci. USA 84:1107-1111).
Other known ADPRT polymerase activity inhibitors include 5-iodo-6-amino-1,2benzopyrone as described in U.S. patent application Ser. No. 600,593, filed Oct. 19, 1990 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and their Metabolites Useful as Cystostatic and Anti-Viral Agents" for use as anti-tumor and anti-viral agents. The cited patent discusses the possibility of using 5-iodo-6-nitroso-1,2-benzopyrone as an anti-tumor or anti-viral agent.
The 6-nitroso-benzopyrones have not been hitherto known or described. The only remotely related compounds found in the literature are 6-nitro-1,2-benzopyrone and 6-amino-1,2-benzopyrone (6-ABP) (J. Pharm. Soc. Jap., 498:615 (1923)) for which, only scarce medicinal evaluation has been reported. In particular, testing was done for sedative and hypnotic effects (J. Pharm. Soc. Japan, 73:351 (1953); Ibid, 74:271 (1954)), hypothermal action (Yakugaku Zasshi, 78:491 (1958)), and antipyretic, hypnotic, hypotensive and adrenolytic action (Ibid, 83:1124 (1963)). No significant application for any of these compounds has been described except for 6-ABP.
2-nitrosobenzamide (Irne-Rasa, K. M. and Koubek, E. (1963) J. Org. Chem. 28:3240-3241), and 4-nitrosobenzamide (Wubbels, G. G., Kalhorn, T. F., Johnson, D. E. and Campbell, D. (1982) J. Org. Chem. 47:4664-4670), have been reported in the chemical literature, but no commercial use of these isomers is known. Neither of these articles suggest the use of nitrosobenzamides as ADPRT inhibitors.
The anti-retroviral and anti-tumorigenic actions of substituted and unsubstituted 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone is the subject of copending U.S. patent applications Ser. No. 585,231 filed on Sep. 21, 1990 entitled "6-Amino-1,2-Benzopyrones Useful for Treatment of Viral Diseases" and Ser. No. 600,593 filed on Oct. 19, 1990 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their Metabolites Useful as Cytostatic and Antiviral Agents", which are incorporated herein by reference.
The precursor molecule, 1,2-benzopyrone (coumarin), was shown to be an inhibitory ligand of adenosinediphosphoribosyl transferase (ADPRT), a DNA-binding nuclear protein present in all mammalian cells (Tseng, et al., (1987) Proc. Nat. Acad, Sci. USA, 84:1107-1111).
Hakam, et al., FEBS Lett., 212:73 (1987) has shown that 6-amino-1,2-benzopyrone (6-ABP) binds specifically to ADRPT at the site that also binds to DNA, indicating that both 6-ABP and DNA compete for the same site on ADPRT. Synthetic ligands of ADPRT inhibit DNA proliferation, particularly in tumorigenic cells, (Kirsten, et al., (1991) Exp. Cell. Res. 193:1-4). Subsequently, these ligands were found to inhibit viral replication and are the subject of the copending U.S. patent application entitled "6-Amino-1-2-Benzopyrones useful for Treatment of Viral Diseases," Ser. No. 585,231, filed on Sep. 21, 1990 which is hereby incorporated by reference.
Retroviral nucleocapsid (NC) proteins and their respective gag precursors from all strains of known retroviruses contain at least one copy of a zinc-binding polypeptide sequence of the type Cys-X.sub.2 -Cys-X.sub.4 -His-X.sub.4 -Cys (CCHC) (Henderson, et al., Biol. Chem. 256:8400-8406 (1981)), i.e., a zinc finger domain. This CCHC sequence is essential for maintaining retroviral infectivity (Gorelick, et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988), Gorelick, et al., J. Virol. 64:3207-3211 (1990)), therefore, it represents an attractive target for retroviral chemotherapy. The HIV-1 gag proteins function by specifically binding to the HIV-1 RNA, anchoring it to the cell membrane for budding or viral particles (Meric, et al., J. Virol. 63:1558-1658 (1989) Gorelick, et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988), Aldovini, et al., J. Virol. 64:1920-1926 (1990), Lever, et al., J. Virol. 63:4085-4087 (1989)). Site-directed mutagenesis studies demonstrated that modification of Cys or His residues results in defective viral RNA packaging and noninfectious viral particles are formed (Aldovini, et al., J. Virol. 64:1920-1926 (1990), Lever, et al., J. Virol. 63:4085-4087 (1989)). The highly abundant nonhistone nuclear protein of eukaryotes, poly(ADP-ribose) polymerase (E.C.2.4.4.30), also contains two CCHC-type zinc fingers located in the basic terminal polypeptide domain, as analyzed by site directed mutagenesis (Gradwohl, et al., Proc. Natl. Sci. USA 87:2990-2992 (1990)).
Published experiments have shown that aromatic C-nitroso ligands of poly (ADPribose) polymerase preferentially destabilize one of the two zinc fingers coincidental with a loss of enzymatic activity but not DNA binding capacity of the enzyme protein (Buki, et al., FEBS Lett. 290:181-185 (1991)). Based on the similarity to results obtained by site-directed mutagenesis (Gradwohl, et al., Proc. Natl. Acad. Sci. USA 87:2990-2992 (1990)), it appears that the primary attack of C-nitroso ligands occurred at zinc finger FI (Buki, et al., FEBS Lett. 290:181-185 (1991)). A selective cytocidal action of the C-nitroso group containing poly (ADP-ribose) polymerase ligands on cancer cells was subsequently discovered (Rice et al., Proc. Natl. Acad. Sci.USA 89:7703-7707.
Based on the coincidental occurrence of the CCHC type zinc fingers in both retroviral NC proteins and in poly(ADP-ribose) polymerase and the observed chemotherapeutic effects of C-nitroso-containing ligands on cancer cells, experiments were initiated to test if the C-nitroso compounds also have antiviral effects on retroviruses containing NC proteins. As described herein experiments testing this hypothesis with the polypeptide corresponding to the N-terminal CCHC zinc finger of HIV-1 NC protein, Zn(HIV1-F1) (South, et al., Am. Chem. Soc. 111:395-396 (1989), South, et al., Biochem. Pharm. 40:123-129 (1990), Summers, et al., Biochemistry 29:329-340 (1990)), intact HIV-1 virions and on the propagation of HIV-1 in human lymphocytes in culture, were performed.